Immune-response is an indispensable defense mechanism against infection for a living body and immune cells are continuously patrolling within a living body in order to rapidly cope with various sources of infection. This feature of the constituting cells to move around without cease is not observed in other living complex systems and has been evolved as a unique feature to the immune system. It is known that, among immune cells, cells such as neutrophils and macrophages function at the early defense against infection whereas T and B lymphocytes induce the antigen-specific immune-response upon recognition of foreign substances through their antigen receptors. Differentiation of such T and B lymphocytes takes place in primary lymphoid organs such as thymus and bone marrows. The differentiated lymphocytes then migrate to a particular compartment in secondary lymphoid organs such as spleen, lymph nodes and Peyer's patches (lymphoid organ in the small intestine), where these lymphocytes induce specific immune-responses by recognizing, via the antigen receptors, antigens recruited from various tissues. In this process, it is considerably crucial for the establishment of immune-response that the lymphocytes migrate into a particular site in secondary lymphoid organs. Although lymphocyte migration has so far been known as being induced by proteins collectively referred to as chemokines of various kinds, the molecular mechanism controlling lymphocyte migration itself has been remained unknown.
Change in cellular polarization and reorganization of cytoskeleton is indispensable for cell migration (Cell 84, 359-369, 1996), both of which have been known to be regulated by low molecular-weight G proteins as Rho, Rac and Cdc42 (Proc. Natl. Acad. Sci. USA 92, 5027-5031, 1995; Science 279, 509-514, 1998; J. Cell Biol. 141, 1147-1157, 1998; Science 287, 1037-1040, 2000). Among these molecules, Rac yields driving force for cell mobility by forming actin-rich lamellipodial protrusion (Science 279, 509-514, 1998; Cell 103, 227-238, 2000). Meanwhile, molecules like CED5, DOCK180 and Myoblast city (MBC) that demonstrate structural homology have been identified for Caenorhabditis elegans, humans and Drosophila melanogaster, which molecules are called CDM family molecules with their acronyms and all of which are thought to be implicated in reorganization of cytoskeleton by functioning in the upstream of Rac (Mol. Cell Biol. 16, 1770-1776, 1996; J. Cell Biol. 138, 589-603, 1997; Nature 392, 501-504, 1998; Genes Dev. 12, 3331-3336, 1998; Genes Dev. 12, 3337-3342, 1998; Nature Cell Biol. 2, 131-136, 2000). Although genetic analysis using mutants has shown that the CED-5 and Myoblast City are crucial for migration of particular types of cells (J. Cell Biol. 138, 589-603, 1997; Nature 392, 501-504, 1998; Nature Cell Biol. 2, 131-136, 2000), in what physiological way the CDM family proteins function in mammals has been left unknown.
It is known than DOCK2 (KIAA0209; DNA Res. 3, 321-329) encodes other CDM family protein member specifically expressed in human haematopoietic cells and that the DOCK2 binds to Rac in 293T kidney cells for activating Rac (Biochem. Biophys. Acta 1452, 179-187, 1999). On the other hand, the present inventors have found upon isolating a novel Hch gene belonging to CDM family from the mouse thymus cDNA library, that the Hch gene product is consisted of 1828 amino acids and SH3 domain is encoded at its N-terminus (Nature, Vol 412, 23 August, 826-831, 2001). Further, it was confirmed in northern blotting using mouse tissues that Hch expression is localized only in the thymus and spleen contrary to the DOCK180 expression which is observed in various organs, and analysis using cell lines provided confirmation that Hch expression is observed in all of T and B cells and macrophages except for two variant T cell lines. Moreover, the present inventors have demonstrated that a significant change in cell morphology as well as enhancement of adhesiveness can be detected by introducing Hch into mutant T cell lines lacking Hch expression. Among the 1828 amino acids encoded by Hch, 1677 amino acids were identical with those of human DOCK2, thus Hch was thought to be mouse homologue of DOCK2, yet physiological function of the DOCK2 remained unrevealed.
Even though immune-response is an indispensable mechanism for a living body, diseases or pathogenic conditions developed as a result of emergence of immune-response, for example autoimmune diseases, graft rejections, GvH, etc., are being focused as a problem which modern medicine is expected to work out for the resolution. For elucidating these diseases or pathogenic conditions at a molecular level or for developing a new therapy for these diseases or pathogenic conditions, molecules have been wanted to be identified that specifically control lymphocyte migration. The subject of the present invention is to identify molecules controlling in a lymphocyte-specific manner migration, and to provide useful animal models for elucidating immune-related diseases or pathogenic conditions such as allergy, autoimmune diseases, GvH or graft rejections at a molecular level, or for developing a new therapy for these diseases or pathogenic conditions.
The present inventors isolated the CDM family DOCK2 (Hch) gene, which is specifically expressed in the immune-system, from a mouse thymus cDNA library, and generated the knockout mice in order to reveal the in vivo function of the gene. DOCK2 knockout mice were born at the expected mendilian ratio without apparent abnormality. However, the numbers of T and B lymphocytes in secondary lymphoid organs such as spleen and lymph nodes were considerably reduced compared to those of wild-type mice. When lymphocytes labeled with fluorescence were intravenously injected into DOCK2 knockout mice for analyzing homing to lymph nodes, homing activity of T and B lymphocytes of the knockout mice was reduced to around 1/10 compared to that of wild-type mice. On the other hand, an efficient homing of lymphocytes to lymph nodes was observed in wild-type mice. These findings thus suggest that homing to secondary lymphoid organs might be impaired in DOCK2 knockout mice owing to an intrinsic defect in lymphocytes.
To address the involvement of DOCK2 molecules in lymphocyte mobility, migration activity in response to various chemokines were compared between the knockout and wild-type mice. No difference was observed between the knockout and wild-type mice as to migration activity of macrophages to chemokines such as MCP-1 or SDF-1. However, contrary to the fact that T and B lymphocytes from wild-type mice actively migrate in response to chemokine stimuli such as with SLC, SDF-1 or BLC, migration of T and B lymphocytes from the knockout mice were significantly impaired. Upon stimulating lymphocytes of knockout mice with chemokines, Rac activation and actin polymerization were observed that peaked at 15 sec. Such responses were disappeared in the knockout mouse lymphocytes. Contrary, no difference was found between the knockout and wild-type mice as to PKB and ERK activations and calcium immigration. These observations demonstrate that DOCK2 specifically controls lymphocyte migration by mediating reorganization of cyteskeleton through activating Rac.
There was no marked abnormality in the differentiation of T and B lymphocytes in the primary lymphoid organs of DOCK2 knockout mice. Peripheral T lymphocytes from the knockout mice, however, were significantly decreased compared to that of the wild-type mice. Since chemokine ELC are known to be involved in emigration of mature thymic T cells from thymus, emigration of these T cells in response to ELC was examined by using thymus organ cultures. The results showed that the emigration efficiency of the knockout mice was reduced to about 1/20 compared to that of the wild-type mice. These results suggest that the emigration defect in mature thymic T cells is responsible for decrease in the peripheral blood T lymphocytes of the knockout mice.
Chemokines such as SLC, ELC and BLC are called “immune-chemokines” and are known to play an essential role in architecture of secondary lymphoid organs. Marked atrophy of lymphoid follicles, straying of lymphocytes into red pulp, and disappearance of marginal-zone B cells were observed in the immunohistological analysis for the spleen of DOCK2 knockout mice. Atrophy of lymphoid follicles was similarly observed in other secondary lymphoid organs such as lymph nodes and Peyer's patches. Besides, marked aberration in distribution of mature thymic T cells in the thymus of the knockout mice was found. It was thus suggested that lymphocytes of DOCK2 knockout mice did not exercise migration activity in response to stimuli with various chemokines, leading to structural abnormality in the immune system.
To study the influence of DOCK2 deficiency on immune-response, mice were immunized with Eα-derived peptide, which is known to bind to MHC class II I-Ab, and the antigen-specific T-cell responses were analyzed. As a result, proliferation response of T cells were observed in an antigen concentration-dependent manner in wild-type mice, whereas such T cell response was not observed in DOCK2 knockout mice. DNP-KLH was also immunized to mice to analyze the KLH-specific antibody production, and the antibody production in DOCK2 knockout mice was observed to have been significantly impaired in DOCK2 knockout mice. These findings thus suggest that primary immune-response was suppressed in DOCK2 knockout mice 7 days after the immunization.
Taking above findings together, it was demonstrated for the first time that DOCK2 is an essential molecule controlling lymphocyte mobility by mediating reorganization of cyteskeleton through activating Rac, and that DOCK2 deficiency largely affect architecture of the immune system and immune-response. These findings are expected to serve for the development of new therapy for immune-related diseases such as allergy, autoimmune diseases and graft rejections by artificially controlling lymphocyte mobility with DOCK2 as a target. The present invention has been completed based on these findings.